Pack - 15 Flashcards
What are the risks of injecting a human donor with animal insulin? (2)
Rejection by the immune system.
Risk of infection.
What is recombinant DNA?
The combining of DNA from two different organisms. (That can be from different species).
What is a transgenic organism? What is is also known as?
- Has had its genome modified (by the process of recombinant DNA)
- Genetically modified organisms.
Why is it that DNA is not only accepted by another species but also functions normally when transferred?
• Genetic code is universal.
Why can proteins still be made from DNA recombinant DNA in another organism?
• The processes of transcription and translation are effectively the same in all organisms.
Describe the 5 stages of making a protein using DNA technology of gene transfer and cloning. Describe each stage.
- Isolation - of DNA fragments that have the gene fir the desired protein.
- Insertion - of DNA fragment into a vector.
- Transformation - transfer of DNA into a suitable host cell.
- Identification - of host cells that have successfully taken up the gene by gene markers.
- growth/cloning of the population.
Give three ways of producing specific gene fragments.
- Conversion of mRNA to cDNA using reverse transcriptase.
- Using restriction endonucleases to cut fragments containing the desired gene.
- A gene machine - based on a known protein structure.
Why does reverse transcriptase have its name? Where is it found?
- It catalyses the production of DNA from RNA (opposite of transcription).
- Retroviruses e.g. HIV
Describe how reverse transcriptase can be used to isolate a gene. (4 steps)
- mRNA is extracted from a cell that produces lots of this mRNA for the desired gene.
- mRNA acts as a template which complementary (cDNA) is formed using reverse transcriptase.
- Hydrolysis of the two strands using an enzyme producing single stranded cDNA
- DNA polymerase is used to from double stranded DNA using the cDNA as template.
Where are restriction endonucleases found and what is their role originally?
Bacteria- to cut up viral DNA
What does a restriction endonuclease do?
Cuts double stranded DNA at a specific base sequence called a recognition sequence.
What is a recognition sequence?
Where restriction endonucleases cut DNA.
What are blunt ends?
When restriction endonucleases cut DNA between two opposite base pairs.
What are sticky ends?
When a restriction endonuclease cuts DNA at different points on each strand leaving exposed single stranded DNA.
What is a palindromic recognition sequence?
The recognition sequence reads the same in both directions.
In 8 steps how can a gene machine produce a gene fragment for a desired gene.
- Base sequence determined. (from amino acid)
- Sequence is checked.
- The computer designs a series of small overlapping single strands of nucleotides - oligonucleotides.
- Each of the oligonucleotides is assembled in an automated process - one nucleotide at a time.
- Oligonucleotides are joined together (no introns)
- Polymerase chain reaction to multiply the gene and construct complementary strand.
- Inserted into a plasmid using sticky ends.
- Genes are checked.
Why is the gene sequence checked before being manufactured by a gene machine?
Biosecurity, biosafety and ethical requirements.
What is the advantage of using a gene machine to produce DNA fragments?
- Any sequence of nucleotides can be produced in a very short amount of time.
- Accurate
Why is it useful for a gene to be free of introns when produced?
• Can be transferred into prokaryotic cells and still be translated.
In what two ways can a DNA fragment be cloned?
- In vivo - transferring the fragments into a host cell using a vector.
- In vitro - PCR
What is the name of the DNA sequence that restriction endonucleases attach to?
Recognition site.
What is the importance of using sticky ends?
Provided the same restriction endonuclease is used, you can combine DNA of one organism with that of any other.
What is the advantage of using sticky ends over blunt ends?
The restriction endonuclease leaves a short sequence of single stranded DNA. This is complementary if the same enzyme is used.
What does DNA ligase do?
Joins the phosphodiester bonds between the two joined sections of DNA.
How two sticky ends from the same enzyme attach?
Complementary base pairing. Then DNA ligase.
What needs to be added to a DNA fragment to be cloned (in vivo) before insertion into a vector?
A promoter - before
A terminator region - after.
What is the purpose of the promoter region of a gene? (2) What does it allow to occur?
- Where RNA polymerase must attach.
- Where transcription factors attach.
- Transcription begins
How does RNA polymerase bind to DNA of a gene?
Nucleotide sequence of the promoter region.
What is the purpose of the terminator region of a gene? What does it terminate?
- RNA polymerase detaches
* Transcription
What is the purpose of inserting target DNA into a vector?
Vector is used to transport the DNA into the host cell.
What is the most common vector used in in vivo cloning?
Plasmids
What is plasmid?
Circular length DNA found in prokaryotic cells. Separate from the main bit of DNA.
What type of gene do plasmids almost always contain?
Antibiotic resistance gene.
Where do restriction endonucleases cut the vector?
In the antibiotic resistance gene.
Why do you need to cut the vector with the same restriction endonuclease as the one used to cut the DNA fragment?
So the sticky ends of the opened up plasmid form complementary base pairs with the sticky ends of the DNA fragment.
How are plasmids joined to DNA fragments permanently?
DNA ligase forms phsophodiester bonds.
What is transformation?
Transferal of recombinant plasmids into the bacterial cells.
Outline the 5 steps of gene transfer and in vivo cloning.
- Isolation of DNA with required gene.
- Insertion into vector using DNA ligase.
- Transformation into host cell.
- Identification of cells that have taken up the recombinant DNA - markers.
- Growth/cloning.
What conditions are used for transformation of the vector into the host cell? (2) How does this work? (1)
- Heat shock
* Calcium ions - make the cell surface membrane permeable so vectors enter.
Why might not all bacterial cells posses the required gene’s DNA after transformation? (3)
- Only about 1% of bacterial cells take up the plasmids when mixed together.
- Some plasmids will have self ligated without DNA fragment.
- Some DNA fragments form loops (“plasmids”) by themselves.
Describe in 4 steps the process of testing whether bacteria have taken up a specific plasmid (with recombinant DNA or NOT) containing the ampicillin resistance gene.
- Bacteria are grown on a medium containing ampicillin.
- Bacterial cells that have taken up the plasmid will be resistant.
- These will survive as they can break down ampicillin.
- The bacterial cells without the plasmids will have no gene and will die.
What is the issue with using only one antibiotic resistance gene to show whether bacteria have taken up a plasmid?
Some bacteria will take up the plasmid without the new gene (self ligated) and so they will survive.
What can be done to identify which bacteria contain the target gene once you have identified which bacteria contain the plasmid (with or without the target gene)?
• Use a marker gene e.g. a second antibiotic resistance gene.
Name three possible types of marker gene.
- Antibiotic resistance (a second one)
- Fluorescent proteine gene
- Enzyme whose action can be identified.
Describe how two antibiotic resistance genes can be used to identify which bacteria have taken up the target gene. (6)
- Cut open the plasmid (restriction endonuclease and sticky ends - same as the one that cut DNA) in the middle of antibiotic resistance A gene.
- DNA forms H-bonds with this plasmid. DNA ligase.
- Gene A no longer works.
- All plasmids contain gene B so grown on a culture of gene B to get rid of those that have taken up no plasmid.
- Replica plate.
- Grow on a culture that contains gene A to see which bacteria die (these are the target bacteria.)
What is replica plating.
Copying colonies of bacteria onto a second plate so when bacteria on one plate die due to an antibiotic these can be identified on the other plate.
A target gene is inserted in the middle of a fluorescent gene in a plasmid. How can this marker be used to identify bacteria with the target gene. Why is this better than two antibiotic resistance genes?
- Those that do not fluoresce have the target gene.
* No need for replica plating as target cells don’t die. Therefore faster.
How is an enzyme gene marker used to identify bacteria that have taken up the required gene?
- Insert target gene into the middle of enzyme gene.
- Enzyme gene doesn’t function.
- Add substrate.
- Colonias that don’t change colour contain the target gene.
What components does the polymerase chain reaction require? (5)
- the DNA fragment to be copied
- taq DNA polymerase
- Primers
- Nucleotides.
- Thermocylcer - computer controlled machine.
What is taq DNA polymerase? Why is it used in the PCR?
- From bacteria in hot springs.
* Doesn’t denature a the high temperatures during PCR.
What is a primer? Why is it necessary in PCR?
- Short sequence of nucleotides that has set bases complementary to those at the end of the DNA fragment.
- DNA polymerase needs a primer to bind to to begin replication. It also prevents the two strands rejoining.
What are the three steps of the PCR? What happens in each? What temp. (approx.)? (3)
- Separation of DNA strands - 95C - Two strands separate - H-bonds break.
- Addition of primers - 55C - primers anneal to the end of base sequence - starting sequence for DNA polymerase - prevent two strands rejoining.
- Synthesis of DNA - 72C - optimum temperature for taq DNA polymerase - add complementary nucleotides to both strands to form two new DNA molecule.
- Repeat
At what rate does the number of DNA strands grow at in PCR?
Exponentially 2^n. N is the tuber of complete cycles.
Give two advantages of in vitro cloning.
- Extremely rapid. (100 billion DAN fragments in hours)
* Does no require living cells - no culturing.
What can PCR be used for that in vivo cloning is not used for? Why?
• Forensics - amplifying DNA at crime scene. Very quick
What is a potential problem with amplifying a tiny amount of DNA found at a crime scene using PCR?
• Contaminating DNA will be greatly amplified as well.
Give 5 advantages of in VIVO cloning.
- Useful when introducing a gene into another organism. e.e once the gene is in plasmid this plasmid can transfer the gene to humans.
- Almost no risk of contamination.
- Very accurate (more than PCR) - mutations are rare.
- Cuts out specific genes - specific gene (not whole DNA sample).
- Produces transformed bacteria that can produce large amounts of the protein.
Why is there little risk of contamination in in vivo cloning?
• Restriction endonuclease match sticky ends to opened up plasmid.
What is a DNA probe?
A short single stranded length of DNA that has some sort of label attached.
What re the two most common types of DNA probe?
- Radioactively labelled - made with nucleotides with a P32 phosphorus isotope.
- Fluorescently labelled probes - fluoresce under certain conditions.
What are DNA probes used for?
To identify particular alleles of genes.
How does a DNA probe identify a particular allele? (4)
- Complementary base sequence to that of the allele.
- Double stranded DNA being tested us separated.
- Separated strands are mixed with probe which binds to complementary bases - DNA hybridisation.
- The site at which the probe binds can be identified by the radioactivity or fluorescence of the probe.
What must be known in order to make a specific DNA probe.
The base sequence of the allele.
What is DNA hybridisation?
When a section of DNA or RNA binds to a single stranded section of DNA with complementary bases.
How does DNA hybridisation work? (3)
- Double stranded DNA heated up.
- Strands Separate
- Cooled - so strands rejoin however if other complementary DNA is present it is just as likely to form base pairs.
How is a specific (mutant) allele for a gene located using DNA hybridisation and DNA probes? (10)
- Determine the base sequence of the allele (genetic library or sequencing)
- DNA fragment produced complementary to allele.
- PCR - amplified.
- Marker attached to make DNA probe.
- DNA from the person being tested is heated (to separate the two strands).
- Cooled in a mixture with DNA probes.
- If the allele is present the DNA probes will binds.
- DNA is washed.
- Remaining DNA will now be fluorescently labelled.
- Dye is detected by shining light onto fragments.
Why is it important to screen individuals who may carry a mutant allele?
If they are heterozygous, they could have homozygous recessive children. Genetic councillors can advise on the potential implications of having children.
How is it possible to test simultaneously for many genetic diseases?
Arrange an array of DNA probes on a glass slide and add a DNA sample.
How can genetic screening be useful in terms of caner?
Detecting for mutations in tumor suppressor genes. If someone inherits one mutated allele they are more at risk of cancer.
What is the benefit of screening mutated tumor suppressor genes?
• Can get early treatment, check for sings of cancer, change lifestyle etc…
How can genetic screening lead to personalised medicine?
For different individuals, some drugs be more effective based on their genotype. Or doctors can work out the dose required to be effective for different individuals.
What is genetic counselling? What does it allow?
Advice and information is given that enable people to make personal decisions e.g. family planning based on genetic disease.
After genetic counselling what decisions could be made?
- Whether to have IVF
* Further tests etc..
How can eugenic screening help treat cancer more effectively? (3)
- Oncogene mutations -determine the type of cancer - most effective treatment.
- Gene changes that predict which patients are more likely to benefit from a treatment.
- Can detect a single cancer cell among millions. Early detection.
What are variable number tandem repeats?
DNA bases that are non coding. Repeated base sequences.
How much of our DNA doesn’t code for a known gene?
95%
Why are VNTRs used in genetic fingerprinting?
Each individual has a unique number and length of VNTRs.
What does gel electrophoresis do?
Separates DNA fragments according to size.
How does gel electrophoresis work?
- DNA fragments placed on agar gel and voltage applied.
- DNA moves to +ve end.
- Resistance of the gel means large DNA fragments move furthest.
- Fragments of different lengths are separated.
- Labelled with probes - e.g. x-ray film placed over final result.
Why does DNA move to the +ve electrode?
• DNA is -ve overall (phosphate)
Outline the 5 steps of making a genetic fingerprint.
- Extraction of DNA from sample (PCR)
- Digestion using restriction endonuclease.
- Separation using gel electrophoresis.
- Hybridisation - DNA probes added.
- Development
Where do the restriction endonucleases cut in genetic fingerprinting?
• In the DNA either side of the target VNTR
What occurs after separation of strands and before the addition of DNA probes in genetic fingerprinting?
Washed with alkali to separate strands.
What is the base sequence of DNA probes complementary to in genetic fingerprinting?
The VNTR base sequence.
How does a computer identify the lengths of DNA on a DNA fingerprint sheet.
Compares them to known lengths of DNA run during gel electrophoresis.
How are two DNA fingerprints compared?
Visually/computer
Give 4 uses of DNA fingerprinting.
- Genetic relationships and variability.
- Forensic Science.
- Medical diagnosis
- Plant and animal breeding.
Explain how DNA fingerprinting is used to identify whether someone is the genetic father of a child. Give some detail. (2)
- Compare Childs genetic fingerprint to its mother and potential father.
- Each band from the child should have a corresponding band on one of the two parents as 50% of their DNA is from each parent.
Explain how DNA fingerprints can give an idea of the genetic diversity of a population.
The more similar the genetic fingerprint the more closely related two organisms are.
If the population has similar fingerprints there is little genetic diversity.
Briefly, how can DNA fingerprinting be used in forensic science.
Take DNA samples from the crime scene. Compare to suspects.
Why might a DNA sample from a crime scene not mean they committed the crime? (4)
- They may have been present but not committed the crime
- May be from a previous occasion.
- DNA may belong to a close relative.
- DNA sample may have been contaminated after the crime. (By the persons DNA or chemicals affecting enzymes)
What must be done before DNA can be used to convict someone?
Probability must be calculated that someone else DNA matches the suspects.
How can genetic fingerprinting be used in medical diagnosis? E.g. Huntington’s where the more repeats of (AGC) at the end of a gene leads to earlier onset of the disease. <30 is unlikely to develop symptoms. >50 early onset likely.
- Fingerprints compared with people who have the disease and those who don’t or those with early onset.
- Probability of developing symptoms can be determined.
How is genetic fingerprinting used in animal breeding?
- Prevent inbreeding
- Identify desirable alleles.
- Establishing a pedigree by identifying of paternity.
